Myeloproliferative neoplasms (MPNs) are hematologic malignancies characterized by gene mutations that promote myeloproliferation and resistance to apoptosis via constitutively active signaling pathways, with Janus kinase 2-signal transducers and the activators of transcription (JAK-STAT) axis as a core part. Chronic inflammation has been described as a pivot for the development and advancement of MPNs from early stage cancer to pronounced bone marrow fibrosis, but there are still unresolved questions regarding this issue. The MPN neutrophils are characterized by upregulation of JAK target genes, they are in a state of activation and with deregulated apoptotic machinery. Deregulated neutrophil apoptotic cell death supports inflammation and steers them towards secondary necrosis or neutrophil extracellular trap (NET) formation, a trigger of inflammation both ways. NETs in proinflammatory bone marrow microenvironment induce hematopoietic precursor proliferation, which has an impact on hematopoietic disorders. In MPNs, neutrophils are primed for NET formation, and even though it seems obvious for NETs to intervene in the disease progression by supporting inflammation, no reliable data are available. We discuss in this review the potential pathophysiological relevance of NET formation in MPNs, with the intention of contributing to a better understanding of how neutrophils and neutrophil clonality can orchestrate the evolution of a pathological microenvironment in MPNs.

Stress is an integral part of life. While acute responses to stress are generally regarded as beneficial in dealing with immediate threats, chronic exposure to threatening stimuli exerts deleterious effects and can be either a contributing or an aggravating factor for many chronic diseases including cancer. Chronic psychological stress has been identified as a significant factor contributing to the development and progression of cancer, but the mechanisms that link chronic stress to cancer remain incompletely understood. Psychological stressors initiate multiple physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic nervous system, and the subsequent changes in immune function. Chronic stress exposure disrupts the homeostatic communication between the neuroendocrine and immune systems, shifting immune signaling toward a proinflammatory state. Stress-induced chronic low-grade inflammation and a decline in immune surveillance are both implicated in cancer development and progression. Conversely, tumor-induced inflammatory cytokines, apart from driving a tumor-supportive inflammatory microenvironment, can also exert their biological actions distantly via circulation and therefore adversely affect the stress response. In this minireview, we summarize the current findings on the relationship between stress and cancer, focusing on the role of inflammation in stress-induced neuroendocrine-immune crosstalk. We also discuss the underlying mechanisms and their potential for cancer treatment and prevention.

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Extracellular adenosine triphosphate (ATP) acts as a signal of disturbed tissue homeostasis and triggers activation of purinergic receptors, thereby affecting various cellular processes such as proliferation, differentiation, and migration. Macrophages migrate toward increasing ATP concentrations and possess P2X7 receptors (P2X7R), which respond to high ATP levels, as well as the ectonucleotidase CD39 that rapidly convert ATP to adenosine monophosphate. Using a mouse model of psychological stress, we recently demonstrated that macrophages substantially support erythropoiesis under chronic stress conditions, but the role of extracellular ATP in stress-induced erythropoiesis remains unknown. Aims: This study was undertaken to investigate the effects of chronic stress on i) extracellular ATP levels, ii) P2X7R expression, and iii) CD39 activity and expression in mouse bone marrow and spleen, and the role of macrophages in stress-induced changes. Methods: Adult male BALB/c mice were subjected to 2h daily restraint stress for 7 consecutive days. Clodronate liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to first restraint procedure, as well as newly recruited macrophages every third day for the duration of the experiment. Single-cell suspensions of bone marrow and spleen samples were obtained from each mouse and 1×106 cells were plated in duplicate. Following the incubation time of 4 h, cell culture supernatants were collected for quantification of extracellular ATP concentrations by the ATP bioluminescence assay. Bone marrow and splenic CD39 cell activity was measured using the malachite green phosphate assay, while the expression levels of CD39 and P2X7R genes in the bone marrow and spleen were determined by quantitative real-time PCR analysis. Results: Chronic exposure to stress markedly increased the extracellular ATP levels in both bone marrow and spleen. Macrophage depletion significantly reduced this stress-induced increase in bone marrow and splenic extracellular ATP concentrations. Changes in extracellular ATP levels among different experimental groups closely resemble those in the number of bone marrow and splenic erythroid progenitor cells previously observed among the same groups (Momčilovic et al. HemaSphere 2022; 6:2469). The increased activity of CD39 was detected in bone marrow and spleen of chronically stressed mice, whereas macrophage depletion abolished this effect of stress. The expression of CD39 and P2X7R were significantly upregulated in the bone marrow and spleen of chronically stressed mice. Treatment with clodronate liposomes did not alter CD39 and P2X7R expression levels under basal conditions but prevented stress-induced elevation of CD39 and P2X7R expression in the bone marrow and spleen. Summary/Conclusion: The obtained results demonstrate a stress-induced increase in extracellular ATP levels, P2X7R expression, and in both CD39 activity and expression within bone marrow and spleen, as well as a significant role of macrophages in stress-induced changes. These findings together with a close resemblance between the stress-induced changes in erythroid progenitor cell number and in extracellular ATP levels suggest that extracellular ATP contributes to erythropoiesis-supportive microenvironment under chronic stress conditions. Keywords: Erythropoieisis, Macrophage

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71-/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes.

Cancer-related anemia (CRA) is a common multifactorial disorder that adversely affects the quality of life and overall prognosis in patients with cancer. Safety concerns associated with the most common CRA treatment options, including intravenous iron therapy and erythropoietic-stimulating agents, have often resulted in no or suboptimal anemia management for many cancer patients. Chronic anemia creates a vital need to restore normal erythropoietic output and therefore activates the mechanisms of stress erythropoiesis (SE). A growing body of evidence demonstrates that bone morphogenetic protein 4 (BMP4) signaling, along with glucocorticoids, erythropoietin, stem cell factor, growth differentiation factor 15 (GDF15) and hypoxia-inducible factors, plays a pivotal role in SE. Nevertheless, a chronic state of SE may lead to ineffective erythropoiesis, characterized by the expansion of erythroid progenitor pool, that largely fails to differentiate and give rise to mature red blood cells, further aggravating CRA. In this review, we summarize the current state of knowledge on the emerging roles for stress erythroid progenitors and activated SE pathways in tumor progression, highlighting the urgent need to suppress ineffective erythropoiesis in cancer patients and develop an optimal treatment strategy as well as a personalized approach to CRA management.

Background: Under steady-state conditions, a population of tissue-resident macrophages within erythroblastic islands is required for erythroid homeostasis. In addition to tissue resident macrophages, newly recruited monocyte-derived macrophages also contribute to erythropoietic niche during stress erythropoiesis. However, recent evidence suggests that the extent of macrophage contribution to stress erythropoiesis is likely to be both tissue- and stress-type dependent. Using a murine model of psychological stress, we have previously shown that chronic stress induces erythropoiesis both in the bone marrow and the spleen, but tissue-specific contribution of macrophages to erythropoiesis during chronic psychological stress remains unknown. Aims: The purpose of this study is to examine the effects of macrophage depletion on bone marrow and splenic erythropoiesis under chronic stress conditions. Methods: Adult male BALB/c mice were subjected to 2h daily restraint stress for 7 consecutive days. Clodronate liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to first restraint procedure, as well as newly recruited macrophages every third day for the duration of the experiment. Mice were randomly assigned to following groups: (1) R - restraint group exposed to daily restraint stress; (2) CLOD + R group, received clodronate liposomes (i.p. 200μl /20g), and subjected to daily restraint; (3) CLOD group, treated with clodronate liposomes only; (4) CTRL + R received control liposomes (i.p. 200μl /20g) and subjected to daily restraint; and (5) control, untreated group. In the bone marrow and spleen number of erythroid progenitors burst forming units-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) were determined using colony asays and CD71/Ter119 profiles of erythroid cells were analyzed by flow citometry. Results: Chronic stress induced a great increase in the number of BFU-E and CFU-E progenitors in the bone marrow and spleen. Macrophage depletion did not significantly alter the number of erythroid progenitors under basal conditions, but completely abolished the effect of repeated stress on BFU-E and CFU-E cells in both the bone marrow and spleen. Flow cytometric analysis of erythroid precursors revealed that macrophage depletion under steady-state conditions substantially reduced the percentage of CD71+/Ter119+ cells in the bone marrow, while the percentage of CD71+/Ter119+ cells in the spleen was not significantly altered. Chronic exposure to daily restraint resulted in considerably increased percentage of CD71+/Ter119+ cells in the bone marrow and spleen, and macrophage depletion fully abrogated the effect of stress on these cells. The treatment with clodronate liposomes under basal conditions increased the percentage of CD71-/Ter119+ cells in the spleen, while the percentage of these cells in the bone marrow remained almost unchanged. The percentage of CD71-/Ter119+ cells was significantly increased in the bone marrow and spleen of chronically stressed mice, and the depletion of macrophages has prevented the effect of stress on these cells. Summary/Conclusion: Under basal conditions, treatment with clodronate liposome induced tissue-specific changes in the percentage of CD71+/Ter119+ and CD71-/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulatory effect of repeated stress on erythroid progenitors and precursors in both the bone marrow and spleen, pointing towards a critical role for macrophages in erythropoiesis under chronic stress conditions.

Anaemia occurs frequently in patients with heart failure and its current treatment lacks clear targets. Emerging evidence suggests that erythroid progenitor cell expansion is an integral part of physiological response to anaemia associated with chronic stress. Understanding the underlying mechanism may provide a novel approach to anaemia management. In this study, we aimed to examine a role for nitric oxide (NO) in the regulation of bone marrow erythroid progenitor response to chronic stress. For this purpose, adult male mice were subjected to 2 h daily restraint stress for 7 or 14 consecutive days. The role of NO was assessed by subcutaneous injection with NG-nitro-l-arginine methyl ester, 30 min prior to each restraint. Chronic exposure to stress resulted in significantly increased number of bone marrow erythroid progenitors, and blockade of NO biosynthesis prior to daily stress completely prevented stress-induced erythroid progenitor cell expansion. Furthermore, chronic stress exposure led to altered expression of neural, endothelial and inducible nitric oxide synthases (NOS) in the bone marrow, both on mRNA and protein level. Decreased expression of neural and endothelial NOS, as well as reduced expression of NF-kappaB/p65 in bone marrow nuclear cell fraction, was accompanied by elevated bone marrow expression of inducible NOS in chronically stressed animals. This is the first study to demonstrate a role for NO in adaptive response of erythroid progenitors to chronic stress. Targeting NO production may be beneficial to improve bone marrow dysfunction and reduced erythroid progenitor cell expansion in chronic heart failure patients.

Adipose tissue (AT) forms depots at different anatomical locations throughout the body, being in subcutaneous and visceral regions, as well as the bone marrow. These ATs differ in the adipocyte functional profile, their insulin sensitivity, adipokines' production, lipolysis, and response to pathologic conditions. Despite the recent advances in lineage tracing, which have demonstrated that individual adipose depots are composed of adipocytes derived from distinct progenitor populations, the cellular and molecular dissection of the adipose clonogenic stem cell niche is still a great challenge. Additional complexity in AT regulation is associated with tumor-induced changes that affect adipocyte phenotype. As an integrative unit of cell differentiation, AT microenvironment regulates various phenotype outcomes of differentiating adipogenic lineages, which consequently may contribute to the neoplastic phenotype manifestations. Particularly interesting is the capacity of AT to impose and support the aberrant potency of stem cells that accompanies tumor development. In this review, we summarize the current findings on the communication between adipocytes and their progenitors with tumor cells, pointing out to the co-existence of healthy and neoplastic stem cell niches developed during tumor evolution. We also discuss tumor-induced adaptations in mature adipocytes and the involvement of alternative differentiation programs.

Macrophage migration inhibitory factor is a well-known proinflammatory cytokine that is released during systemic stress response. Although MIF can affect erythrocyte production, the role of this cytokine in stress-induced erythropoiesis is completely unknown. To extend our previous findings showing that chronic psychological stress stimulates extramedullary erythropoiesis, here we examined whether MIF is involved in the control of stress-induced erythropoietic response. Adult male C57BL/6 wild-type (WT) and MIF-KO (knock-out) mice were subjected to 2-h daily restraint stress for either 7 or 14 consecutive days. The number of erythroid progenitors and CD71/Ter119 profile of erythroid precursors were analyzed in the bone marrow and spleen. Additionally, MIF protein expression was assessed in WT mice. Our results demonstrated that chronic restraint stress enhanced the number of both erythroid progenitors and precursors in the spleen. Stress-induced increase in the number of splenic late erythroid progenitors as well as in the percentage of CD71+Ter119+-double-positive precursors was significantly more pronounced in MIF-KO mice compared to WT animals. Furthermore, repeatedly stressed WT animals demonstrated an augmented MIF expression in the spleen. Unlike the spleen, the bone marrow of chronically stressed WT mice exhibited less prominent changes in erythropoietic stress response and no significant alteration in MIF expression. In addition, MIF deficiency did not influence the bone marrow erythropoiesis in stressed animals. These findings suggest that MIF regulates extramedullary erythropoiesis by inhibiting an overexpansion of splenic immature erythroid cells during chronic stress and indicate a novel role for this cytokine under chronic stress conditions.

Stress evokes an integrated neuroendocrine response perturbing the homeostasis of different physiological systems. In contrast to well established physiologica linteractions between neuroendocrine and immune systems during chronic stress, there has been relatively little information on the effects of psychological stress on erythroid cells. Since stress-induced erythropoiesis occurs predominantly in the spleen, in the current study, we investigated the influence of chronic psychological stress on splenic erythroid progenitors and examined a role of glucocorticoid receptor (GR) in observed effect using a mouse model of restraint. The adult male mice were subjected to 2 hours daily restraint stress for 7 or 14 consecutive days and the role of GR in erythropoietic response to stress was assessed by pretreatment of mice with GR antagonist mifepristone 60 min prior to restraint. The results showed that chronic restraint stress induced an increase in spleen weight as well as in the cellularity of red pulp, as compared to controls. Furthermore, 7 and 14 days of restraint stress resulted in markedly increased number of both splenic early (BFU-E) and late (CFU-E) erythroid progenitors. Blockade of GR with mifepristone did not affect the number of BFU-E in stressed mice, but it completely abolished the effect of repeated psychological stress on CFU-E cells. Additionally, plasma corticosterone concentration was enhanced whereas the GR expression was significantly decreased within splenic red pulp after one and two weeks of stress exposure. Obtained findings suggest for the first time an indispensable role for GR in the expansion of CFU-E progenitors in the spleen under conditions of chronic psychological stress.

The aim of our study was to investigate the appearance, density and distribution of ghrelin cells and GHS-R1a and GHS-R1b in the human stomach and duodenum during prenatal and early postnatal development. We examined chromogranin-A and ghrelin cells in duodenum, and GHS-R1a and GHS-R1b expression in stomach and duodenum by immunohistochemistry in embryos, fetuses, and infants. Chromogranin-A and ghrelin cells were identified in the duodenum at weeks 10 and 11 of gestation. Ghrelin cells were detected individually or clustered within the base of duodenal crypts and villi during the first trimester, while they were presented separately within the basal and apical parts of crypts and villi during the second and third trimesters. Ghrelin cells were the most numerous during the first (∼11%) and third (∼10%) trimesters of gestation development. GHS-R1a and GHS-R1b were detected at 11 and 16 weeks of gestation, showed the highest level of expression in Brunner's gland and in lower parts of duodenal crypts and villi during the second trimester in antrum, and during the third trimester in corpus and duodenum. Our findings demonstrated for the first time abundant duodenal expression of ghrelin cells and ghrelin receptors during human prenatal development indicating a role of ghrelin in the regulation of growth and differentiation of human gastrointestinal tract.

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4-dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.

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